Many proteins are valuable as therapeutic agents. Protein therapeutics are often produced using recombinant DNA technology, which can enable production of higher amounts of protein than can be isolated from naturally-occurring sources, and which avoids contamination that often occurs with proteins isolated from naturally-occurring sources.
Proper folding of a protein is essential to the normal functioning of the protein. Improperly folded proteins are believed to contribute to the pathology of several diseases, including Alzheimer's disease, bovine spongiform encephalopathy (BSE, or “mad cow” disease) and human Creutzfeldt-Jakob disease (CJD), and Parkinson's disease; these diseases serve to illustrate the importance of proper protein folding.
Proteins of therapeutic value in humans can be expressed in bacteria, yeast, and other microorganisms. While large amounts of proteins can be produced in such systems, the proteins are often misfolded, and often aggregate together in large clumps called inclusion bodies. The proteins cannot be used in the misfolded, aggregated state. Accordingly, methods of disaggregating and properly refolding such proteins have been the subject of much investigation.
One method of refolding proteins uses high pressure on solutions of proteins in order to disaggregate, unfold, and properly refold proteins. Such methods are described in U.S. Pat. No. 6,489,450, U.S. Pat. No. 7,064,192, U.S. Patent Application Publication No. 2004/0038333, and International Patent Application WO 02/062827. Those disclosures indicated that certain high-pressure treatments of aggregated proteins or misfolded proteins resulted in recovery of disaggregated protein retaining biological activity (i.e., the protein was properly folded, as is required for biological activity) in good yields. U.S. Pat. No. 6,489,450, U.S. Pat. No. 7,064,192, U.S. 2004/0038333, and WO 02/062827 are incorporated by reference herein in their entireties.
Certain devices have also been developed which are particularly suitable for refolding of proteins under high pressure; see International Patent Application Publication No. WO 2007/062174, which is incorporated by reference herein in its entirety.
Several monoclonal antibodies are currently in use as therapeutic agents, for example, Herceptin® (Trastuzumab) (Herceptin® is a registered trademark of Genentech, Inc., South San Francisco, Calif., for a monoclonal antibody useful in treating breast cancer) and Remicade® (Infliximab) (Remicade® is a registered trademark of Centocor, Inc., Malvern, Pa., for a monoclonal antibody useful in treating inflammatory disorders involving the immune system such as rheumatoid arthritis). Unfortunately, some of the most widely used processing steps for monoclonal antibody production, such as Protein A/G affinity purification and/or viral inactivation steps, require use of solutions at pH levels as low as approximately pH 3.0 during typical pharmaceutical protein manufacturing (Ejima et al., Proteins, 66:954-62 (2007)). Monoclonal antibodies readily aggregate during treatment at pH 3.0, possibly due to destabilization of the Fc domain. These aggregates can be difficult to remove and result in increased production costs. See, Thommes, J. and M. Etzel, Biotechnology Progress 23(1): 42-45 (2007) for a discussion of these issues. High pressure refolding provides a viable method for alleviating aggregation of monoclonal antibodies induced by manufacturing processes.
The effect of aggregate formation conditions on the pressure-modulated refolding yield is currently unknown. An earlier report (St. John, R. J., J. F. Carpenter, et al., Journal of Biological Chemistry 276(50): 46856-46863 (2001)) showed that the refolding yields of recombinant human growth hormone from two different insoluble aggregates contained different secondary structures and resulted in different refolding kinetics and yields. Thus, protein aggregates produced by different stresses exhibited different refolding behaviors. Consequently, the specific conditions required for refolding mAb aggregates formed after incubation at pH 3.0 may be unpredictable.
The instant invention provides methods useful in refolding monoclonal antibody aggregates produced after exposure to low pH, for example, approximately pH 3.0, as well as preparations containing such monoclonal antibody aggregates.